The study of the recognition or lack of recognition of foreign antigen by a host organism has proceeded in many different directions. Understanding of the field presumes some understanding of both basic immunology, and protein chemistry.
The immune system is constantly at war, fighting viruses, bacteria, and other pathogens that try to invade the body. In this war, it uses a diverse range of effectors in order to deal with the threat to health posed by an equally diverse range of infectious organisms that are prevalent in the environment. For example, T-lymphocytes play a central role in the induction and regulation of the immune response and in the execution of immunological functions. These cells are particularly important in tumor rejection and in fighting viral infections.
However, antigen recognition by the T-lymphocytes is restricted by cell-surface glycoproteins encoded by the highly polymorphic genes of the major histocompatibility complex (MHC) molecules. This discrimination requires a T cell specific for a particular combination of an HLA molecule and a peptide rather than the intact foreign antigen itself. If a specific T cell is not present, there is no T cell response even if its partner complex is present. Similarly, there is no response if the specific complex is absent, but the T cell is present. Much work has focused on the mechanisms by which proteins are processed into the HLA binding peptide. See, in this regard, Cresswell, Nature 343: 593-594 (1990); Aichinger, et al. Biochemical Society Trans, 23: 657-659, (1995); Fremont et al., Science 257: 919 (1992); and Lanzavecchia, Science 260:937-943 (1993); Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257: 964 (1992).
MHC class I molecules are expressed by almost all nucleated cells of the body and, in the main, present peptides derived from intracellular proteins to cytotoxic T cells expressing the CD8 co-receptor. Activation of the cytotoxic T cell, in turn, results in the destruction of the target cells by apoptosis induced by perforin/granzyme and/or Fas ligand.
In the case of MHC class I molecules, the peptide fragments usually contain from 8 to 11 amino acids and are generated inside the antigen presenting cells by a complex machinery involving proteases, peptide transporters and molecular chaperones. See Roitt, et al. Immunology (Mosby-Year Book Europe, 1993).
Although intact proteins need to be processed to generate antigenic peptide, soluble peptides are also known to directly bind to small fractions of empty MHC class I or II molecules present on cell surfaces. See in this regard, Braciale & Braciale, Immunology Today, 12(4): 124-129 (1991); Elliot, Immunology Today 12(11): 386-388 (1991).
MHC class II molecules are expressed on a more limited set of antigen presenting cells, exemplified by B-cells, T-cells themselves, macrophages, and dendritic cells. These molecules present peptides in a way which is similar to class I molecules, except that the peptide is generally derived from an exogenous protein from the intercellular environment (e.g., bacteria). Class II molecules present their captured peptide to helper T cells expressing the CD4 co-receptor molecule and their activation generally results in the secretion of cytokines.
Thus, specific T cell immunity is controlled by two selective and independent binding events: first, binding of the peptide fragments of the antigens by the MHC class molecules, and second, binding of the resulting complexes by the clonotypic antigen receptors of the T cell. See, in this regard, Ada, Immunology and Cell Biology 72:447-454 (1994).
Tumor antigens are characteristic of tumor tissue and thus may be considered tissue specific. Tumor antigens result from alterations that frequently occur in malignant transformation of normal tissue. The alteration may be quantitative in that a particular normal antigen may decrease or increase in concentration. Such normal antigens that have increased concentration in tumors are generally referred to as "Tumor Associated Antigens (TAAs)" Antigenic alteration may also be qualitative in that a new antigen, foreign to the host, may appear. These are termed "Tumor-Specific Antigens (TSAs") and may be present as new cell-surface structures or as new intracellular structures in the cytoplasm or nucleus.
Tumor specific antigens were first clearly demonstrated in mice that had been immunized with cells from a methylcholanthrene-induced sarcoma taken from syngeneic mice. These molecules were "recognized" by T cells in the recipient animal, and provoked a cytolytic T cell ("CTL" hereafter) response with lysis of the transplanted cells. The antigens expressed by the tumors and which elicited the T cell response were found to be different for each tumor. See Prehn, et al., J. Natl. Canc. Inst. 18: 769-778 (1957); Klein et al., Cancer Res. 20: 1561-1572 (1960); Gross, Cancer Res. 3: 326-333 (1943), Basombrio, Cancer Res. 30: 2458-2462 (1970) for general teachings on inducing tumors with chemical carcinogens and differences in cell surface antigens. This class of antigens has come to be known as "Tumor Specific Transplantation Antigens" or "TSTAs". Following the observation of the presentation of such antigens when induced by chemical carcinogens, similar results were obtained when tumors were induced in vitro via ultraviolet radiation. See Kripke, J. Natl. Canc. Inst. 53: 333-1336 (1974) Prehn, R. T., and Main, J. M, Journal of Natl. Cancer Inst. 18:769 (1974).
While T cell mediated immune responses were observed for the types of tumor described supra, spontaneous tumors were thought to be generally non-immunogenic. These were therefore believed not to present antigens which provoked a response to the tumor in the tumor carrying subject. See Hewitt, et al., Brit. J. Cancer 33: 241-259 (1976).
The family of tum.sup.- antigen presenting cell lines are immunogenic variants obtained by mutagenesis of mouse tumor cells or cell lines, as described by Boon et al., J. Exp. Med. 152: 1184-1193 (1980), the disclosure of which is incorporated by reference. To elaborate, tum.sup.- antigens are obtained by mutating tumor cells which do not generate an immune response in syngeneic mice and will form tumors (i.e., "tum.sup.+ " cells). When these tum.sup.+ cells are mutagenized, they are rejected by syngeneic mice, and fail to form tumors (thus "tum.sup.- "). See Boon et al., Proc. Natl. Acad. Sci. USA 74: 272 (1977), the disclosure of which is incorporated by reference. Many tumor types have been shown to exhibit this phenomenon. See, e.g., Frost et al., Cancer Res. 43: 125 (1983).
It appears that tum.sup.- variants fail to form progressive tumors because they elicit an immune rejection process. The evidence in favor of this hypothesis includes the ability of "tum.sup.- " variants of tumors, i.e., those which do not normally form tumors, to do so in mice with immune systems suppressed by sublethal irradiation, Van Pel et al., Proc. Natl, Acad. Sci. USA 76: 5282-5285 (1979); and the observation that intraperitoneally injected tum.sup.- cells of mastocytoma P815 multiply exponentially for 12-15 days, and then are eliminated in only a few days in the midst of an influx of lymphocytes and macrophage (Uyttenhove et al., J. Exp. Med. 152: 1175-1183 (1980)). Further evidence includes the observation that mice acquire an immune memory which permits them to resist subsequent challenge to the same tum.sup.- variant, even when immunosuppressive amounts of radiation are administered with the following challenge of cells (Boon et al., Proc. Natl, Acad. Sci. USA 74: 272-275 (1977); Van Pel et al., supra; Uyttenhove et al., supra).
Later research found that when spontaneous tumors were subjected to mutagenesis, immunogenic variants were produced which did generate a response. Indeed, these variants were able to elicit an immune protective response against the original tumor. See Van Pel et al., J. Exp. Med. 157: 1992-2001 (1983). Thus, it has been shown that it is possible to elicit presentation of a so-called "TRA" in a tumor which is a target for a syngeneic rejection response. Similar results have been obtained when foreign genes have been transfected into spontaneous tumors. See Fearson et al., Cancer Res. 48: 2975-1980 (1988) in this regard.
The extent to which these antigens have been studied, has been via cytolytic T cell characterization studies, in vitro i.e., the study of the identification of the antigen by a particular cytolytic T cells ("CTL" hereafter) subset. The subset proliferates upon recognition of the presented tumor rejection antigen, and the cells presenting the antigen are lysed.
Characterization studies have identified CTL clones which specifically lyse cells expressing the antigens. Examples of this work may be found in Levy et al., Adv. Cancer Res. 24: 1-59 (1977); Boon et al., J. Exp. Med. 152: 1184-1193 (1980); Brunner et al., J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 124: 1627-1634 (1980); Maryanski et al., Eur. J. Immunol. 12: 406-412 (1982); Palladino et al., Canc. Res. 47: 5074-5079 (1987).
A tumor exemplary of the subject matter described supra is known as P815. See DePlaen et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988); Szikora et al., EMBO J 9: 1041-1050 (1990), and Sibille et al., J. Exp. Med. 172: 35-45 (1990), the disclosures of which are incorporated by reference. The P815 tumor is a mastocytoma, induced in a DBA/2 mouse with methylcholanthrene and cultured as both an in vitro tumor and a cell line. The P815 line has generated many tum.sup.- variants following mutagenesis, including variants referred to as P91A (DePlaen, supra), 35B (Szikora, supra), and P198 (Sibille, supra).
Hence, with reference to the literature, a cell line can be tum.sup.+, such as the line referred to as "P1", and can be provoked to produce tum.sup.- variants. Since the tum.sup.- phenotype differs from that of the parent cell line, one expects a difference in the DNA of tum.sup.- cell lines as compared to their tum.sup.+ parental lines, and this difference can be exploited to locate the gene of interest in tum.sup.- cells. As a result, it was found that genes of tum.sup.- variants such as P91A, 35B and P198 differ from their normal alleles by point mutations in the coding regions of the gene. See Szikora and Sibille, supra, and Lurquin et al., Cell 58: 293-303 (1989). This has proved not to be the case with the "Tumor Rejection Antigens TRAs" of this invention. These papers also demonstrated that peptide derived from the tum.sup.- antigen are presented by the H-2 molecule for recognition by CTLs. P91A is presented by L.sup.d, P35 by D.sup.d and P198 by K.sup.d.
Human melanoma cells also bear antigens that are recognized by autologous CD8.sup.+ cytolytic T cells, which can be derived from blood lymphocytes or from tumor-infiltrating lymphocytes. In PCT application PCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, and incorporated by reference, a family of genes is disclosed, which are processed into peptides which, in turn, are expressed on cell surfaces, which can lead to lysis of the tumor cells by specific cytolytic T lymphocytes. The genes are said to code for "tumor rejection antigen precursors" or "TRAP" molecules, and the peptides derived therefrom are referred to as "tumor rejection antigens" or "TRAs". See Traversari et al., Immunogenetics 35: 145 (1992); van der Bruggen et al., Science 254: 1643 (1991), for further information on this family of genes. Also, see U.S. Pat. No. 5,342,774, incorporated by reference in its entirety, which discloses the "MAGE" family of tumor rejection antigen precursors.
The tum.sup.- antigens are only present after the tumor cells are mutagenized. In contrast, tumor rejection antigens--and this is a key distinction--are present on cells of a given tumor without mutagenesis.
U.S. Pat. No. 5,405,940, the disclosure of which is incorporated by reference, contemplates isolated nonapeptides derived from MAGE genes. In this patent, it is explained that the MAGE-1 gene codes for a TRAP which is processed to nonapeptides that are presented by HLA-A1 molecules. According to this patent, the nonapeptides are derived from expression products of the MAGE gene family. The resulting complexes are identified by cytolytic T cells, which can be used in diagnostics or therapeutically. The nonapeptides which bind to HLA-A1 follow a "rule" for binding in that a motif is satisfied. In this regard, see e.g. PCT/US93/07421; Falk et al., Nature 351: 290-296 (1991); Engelhard, Ann Rev. Immunol. 12: 181-207 (1994); Ruppert et al., Cell 74: 929-937 (1993); Rotzschke et al., Nature 348: 252-254 (1990); Bjorkman et al., Nature 329: 512-518 (1987); Traversari et al., J. Exp. Med. 176: 1453-1457 (1992). These references teach that given the known specificity of particular peptide for particular HLA molecules, one should expect a particular peptide to bind to at least one HLA molecule.
A cursory review of the development of the field may be found in Barinaga, "Getting Some `Backbone`: How MHC Binds Peptide", Science 257: 880 (1992); also, see Fremont et al., Science 257: 919 (1992); Matsumura et al., Science 257: 927 (1992); Latron et al., Science 257: 964 (1992). These papers generally point to a preference that the peptide which binds to an MHC/HLA molecule be nine amino acids long (a "nonapeptide"), and to the importance of two so-called anchor residues (most commonly the second and ninth residues of the nonapeptide).
Studies on the MAGE family of genes have now revealed that particular peptides are in fact presented on the surface of tumor cells, and that the presentation of the peptide requires that the presenting molecule be a specific HLA molecule. Complexes of the MAGE-1 tumor rejection antigen (the "TRA" or nonapeptide") and the HLA leads to lysis of the cell presenting it by CTL. This observation has both diagnostic and therapeutic implications, as discussed herein.
It has also been found that, when comparing homologous regions of various MAGE genes to the region of the MAGE-1 gene coding for the relevant nonapeptide, there is a great deal of homology. Homologous peptides can be used for various purposes which includes their use as immunogens, either alone or coupled to carrier peptide. The peptides are of sufficient size to constitute an antigenic epitope, and the antibodies generated thereto may then be used to identify the peptide, either as it exists alone, or as part of a larger polypeptide.
The nonapeptides may also be used as agents to identify various HLA subtypes on the surface of tumor cells, such as melanomas. Via this ability they may serve either as diagnostic markers, or as therapeutic agents. These features are discussed infra.
A second class of antigens represents differentiation antigens encoded by genes that are expressed in melanoma and in normal melanoctytes. Antigens derived from tyrosinase are exemplary of this class.
It is noteworthy that while the prior art peptides might arguably find use as therapeutics or in diagnostics, their contemplated use is short lived in view of their rapid degradation by peptidase activity.